目的:制备纳米粒载体DMP,对其进行表征的测定,研究DMP介导的Bcl-xl siRNA的抗肿瘤活性。方法:采用MTT法对纳米粒的细胞毒性进行测定;通过Q-PCR法检测mRNA的含量,检测DMP递送Bcl-xlsiRNA入C26细胞的基因沉默效果;通过MTT法研究DMP/si RNA...目的:制备纳米粒载体DMP,对其进行表征的测定,研究DMP介导的Bcl-xl siRNA的抗肿瘤活性。方法:采用MTT法对纳米粒的细胞毒性进行测定;通过Q-PCR法检测mRNA的含量,检测DMP递送Bcl-xlsiRNA入C26细胞的基因沉默效果;通过MTT法研究DMP/si RNA对C26细胞的抗肿瘤效果,测定其对C26细胞的肿瘤抑制作用;用克隆形成实验进一步证明DMP/siRNA能够抑制C26细胞生长,具有显著的抗肿瘤活性;采用碘化丙啶(PI)染色法流式细胞术来分析经过DMP/siBcl-xl治疗的C26细胞的凋亡率。结果:纳米粒具有良好的粒径和电位以及较低的细胞毒性,其IC50为3.7μg﹒mL^(-1)。Q-PCR结果显示DMP/siBcl-xl有效地降低了Bcl-xl信使RNA的水平;MTT结果显示DMP/siBcl-xl(50 n M和100 n M)的生存率分别为69.6%±3.3%,56.3%±1.9%,低于DMP组;克隆形成实验表明DMP/siBcl-xl能够抑制C26细胞的增殖,具有显著的抗肿瘤活性;凋亡实验结果显示DMP/siBcl-xl的细胞凋亡率为33.0%±3.8%,与对照组、DMP组、DMP/Scramble siRNA组比较,细胞凋亡率显著地增加了。结论:纳米粒DMP具有良好的粒径和电位,并且具有较低的细胞毒性。DMP/siBcl-xl能够沉默Bcl-xl基因,引发C26细胞的凋亡,进一步实现抑制C26细胞生长的治疗作用。实验表明纳米粒DMP能够作为有效的载体递送siRNA用于结肠癌的治疗。展开更多
目的观察胶质细胞Slit1对脊髓背根节(DRG)神经元突起生长的影响。方法构建大鼠Slit1siRNA,转染至大鼠原代DRG卫星胶质细胞。利用实时荧光定量-聚合酶链反应(RT-PCR)、Western blot技术检测Slit1 m RNA和蛋白的表达情况;将siRNA-Slit1转...目的观察胶质细胞Slit1对脊髓背根节(DRG)神经元突起生长的影响。方法构建大鼠Slit1siRNA,转染至大鼠原代DRG卫星胶质细胞。利用实时荧光定量-聚合酶链反应(RT-PCR)、Western blot技术检测Slit1 m RNA和蛋白的表达情况;将siRNA-Slit1转染成功的胶质细胞与大鼠原代DRG神经元共培养48 h,显微镜下观察神经元突起生长情况。结果 (1)与阴性对照组比较,siRNA-Slit1组胶质细胞Slit1 m RNA的表达量明显降低,差异具有统计学意义(P<0.05);空白对照组与阴性对照组比较差异无统计学意义(P>0.05);(2)siRNA-Slit1组Slit1蛋白的表达量明显低于阴性对照组,差异具有显著统计学意义(P<0.01),而空白对照组与阴性对照组间比较差异无统计学意义(P>0.05);(3)siRNA-Slit1组神经元突起长度明显短于阴性对照组,差异具有显著统计学意义(P<0.01)。结论 Slit1是诱导神经元突起生长的重要导向分子,可促进离体DRG神经元突起的生长。展开更多
Purpose:To investigate the effect of small interfering RNA (siRNA)targeting VEGF of retinal pigment epithelium (RPE) cells on the growth activity of human retinal vascular endothelial cells (RECs) under a co-culture s...Purpose:To investigate the effect of small interfering RNA (siRNA)targeting VEGF of retinal pigment epithelium (RPE) cells on the growth activity of human retinal vascular endothelial cells (RECs) under a co-culture system.Methods:By applying the vector.(pGPU6)-based siRNA plasmid gene silence system,we specifically silenced VEGF expression of RPE cells (ARPE-19) through plasmid (pG-PU6-VEGFA-siRNA)transfection.Reverse transcription polymerase chain reaction(RT-PCR) was applied for selecting the most efficient siRNA segment among three pGPU6-VEGF-siRNA groups (siRNA-1,siRNA-2 and siRNA-3).Treated RPE cells were co-cultured with RECs in a co-culture system made up of a 24-well culture plate and transwell inserts assembled inside During 7-day culture period,the growth ca-pacity of RECs were observed and tested in the form of cell counting assay.Three groups were established in this study:RPE cells transfected with pGPU6-VEGF-siRNA and co-cultured with RECs (group A),RPE cells transfected with siR-NA null vector and co-cultured with RECs (group B),and RECs cultured alone (group C).Results:After transfection,VEGF expression levels of RPE cells in three pGPU6-VEGF-siRNA groups (siRNA-1,siR-NA-2 and siRNA-3)evaluated by RT-PCR were 2.56 ±0.45,1.17 ±0.38 and 4.39 ±0.51,respectively (n =10).siRNA-2 was selected as the foremost segment for transfection (P < 0.05,SNK-q test).During the 7-day co-culture period,the influence upon the growth of RECs was observed.Growth curve of RECs under co-culture showed a lower growth rate in group A than in group B (P <0.05,dunnett's test),but no significant difference between group A and group C was noted (P>0.05,dunnett's test).RECs in group A proliferated muchfaster during the first four days post-transfection.Conclusion:Delivery of siRNA targeting VEGF plays an efficient role in down-regulating VEGF expression in RPE cells,therefore modulating the growth activity of RECs under a co-culture system in vitro.The application of this technique may provide novel evidence for the prevention and treatment of retinal neovascularization diseases.展开更多
文摘目的:制备纳米粒载体DMP,对其进行表征的测定,研究DMP介导的Bcl-xl siRNA的抗肿瘤活性。方法:采用MTT法对纳米粒的细胞毒性进行测定;通过Q-PCR法检测mRNA的含量,检测DMP递送Bcl-xlsiRNA入C26细胞的基因沉默效果;通过MTT法研究DMP/si RNA对C26细胞的抗肿瘤效果,测定其对C26细胞的肿瘤抑制作用;用克隆形成实验进一步证明DMP/siRNA能够抑制C26细胞生长,具有显著的抗肿瘤活性;采用碘化丙啶(PI)染色法流式细胞术来分析经过DMP/siBcl-xl治疗的C26细胞的凋亡率。结果:纳米粒具有良好的粒径和电位以及较低的细胞毒性,其IC50为3.7μg﹒mL^(-1)。Q-PCR结果显示DMP/siBcl-xl有效地降低了Bcl-xl信使RNA的水平;MTT结果显示DMP/siBcl-xl(50 n M和100 n M)的生存率分别为69.6%±3.3%,56.3%±1.9%,低于DMP组;克隆形成实验表明DMP/siBcl-xl能够抑制C26细胞的增殖,具有显著的抗肿瘤活性;凋亡实验结果显示DMP/siBcl-xl的细胞凋亡率为33.0%±3.8%,与对照组、DMP组、DMP/Scramble siRNA组比较,细胞凋亡率显著地增加了。结论:纳米粒DMP具有良好的粒径和电位,并且具有较低的细胞毒性。DMP/siBcl-xl能够沉默Bcl-xl基因,引发C26细胞的凋亡,进一步实现抑制C26细胞生长的治疗作用。实验表明纳米粒DMP能够作为有效的载体递送siRNA用于结肠癌的治疗。
文摘目的观察胶质细胞Slit1对脊髓背根节(DRG)神经元突起生长的影响。方法构建大鼠Slit1siRNA,转染至大鼠原代DRG卫星胶质细胞。利用实时荧光定量-聚合酶链反应(RT-PCR)、Western blot技术检测Slit1 m RNA和蛋白的表达情况;将siRNA-Slit1转染成功的胶质细胞与大鼠原代DRG神经元共培养48 h,显微镜下观察神经元突起生长情况。结果 (1)与阴性对照组比较,siRNA-Slit1组胶质细胞Slit1 m RNA的表达量明显降低,差异具有统计学意义(P<0.05);空白对照组与阴性对照组比较差异无统计学意义(P>0.05);(2)siRNA-Slit1组Slit1蛋白的表达量明显低于阴性对照组,差异具有显著统计学意义(P<0.01),而空白对照组与阴性对照组间比较差异无统计学意义(P>0.05);(3)siRNA-Slit1组神经元突起长度明显短于阴性对照组,差异具有显著统计学意义(P<0.01)。结论 Slit1是诱导神经元突起生长的重要导向分子,可促进离体DRG神经元突起的生长。
文摘Purpose:To investigate the effect of small interfering RNA (siRNA)targeting VEGF of retinal pigment epithelium (RPE) cells on the growth activity of human retinal vascular endothelial cells (RECs) under a co-culture system.Methods:By applying the vector.(pGPU6)-based siRNA plasmid gene silence system,we specifically silenced VEGF expression of RPE cells (ARPE-19) through plasmid (pG-PU6-VEGFA-siRNA)transfection.Reverse transcription polymerase chain reaction(RT-PCR) was applied for selecting the most efficient siRNA segment among three pGPU6-VEGF-siRNA groups (siRNA-1,siRNA-2 and siRNA-3).Treated RPE cells were co-cultured with RECs in a co-culture system made up of a 24-well culture plate and transwell inserts assembled inside During 7-day culture period,the growth ca-pacity of RECs were observed and tested in the form of cell counting assay.Three groups were established in this study:RPE cells transfected with pGPU6-VEGF-siRNA and co-cultured with RECs (group A),RPE cells transfected with siR-NA null vector and co-cultured with RECs (group B),and RECs cultured alone (group C).Results:After transfection,VEGF expression levels of RPE cells in three pGPU6-VEGF-siRNA groups (siRNA-1,siR-NA-2 and siRNA-3)evaluated by RT-PCR were 2.56 ±0.45,1.17 ±0.38 and 4.39 ±0.51,respectively (n =10).siRNA-2 was selected as the foremost segment for transfection (P < 0.05,SNK-q test).During the 7-day co-culture period,the influence upon the growth of RECs was observed.Growth curve of RECs under co-culture showed a lower growth rate in group A than in group B (P <0.05,dunnett's test),but no significant difference between group A and group C was noted (P>0.05,dunnett's test).RECs in group A proliferated muchfaster during the first four days post-transfection.Conclusion:Delivery of siRNA targeting VEGF plays an efficient role in down-regulating VEGF expression in RPE cells,therefore modulating the growth activity of RECs under a co-culture system in vitro.The application of this technique may provide novel evidence for the prevention and treatment of retinal neovascularization diseases.
